The role of ADP-Ribose as mRNA cap during cellular starvation

• Applicant: Dr. Roko Zaja
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 534090377

ADP-ribosylation is a highly conserved covalent modification in which an ADP-ribose (ADPr) moiety is transferred from the cofactor NAD+ to target molecules. While proteins have traditionally been considered the main targets of ADP-ribosylation, recent studies have identified also RNA as substrates for mammalian ADP-ribosyltransferases. Our research has confirmed that specific enzymes can modify single-stranded RNA with ADPr at the 5`-end and demonstrated that an ADPr cap protects RNA from degradation by 5'-exonucleases. However, the function of RNA ADP-ribosylation in cells is not understood. We have observed that ADP-ribose is a highly dynamic mRNA cap in cells, which becomes more prevalent during nutrient starvation with unknown function. We have now developed a sequencing technique to analyse ADP-ribosylated mRNA in cells. In preliminary experiments, we have identified specific mRNAs related to regulation of translation and ATP production that undergo rapid ADP-ribosylation upon amino acid starvation. The main objective of this proposal is to first identify the mRNAs that are ADP-ribosylated in response to starvation and subsequently we will investigate localization, stability and translation of specific mRNAs. Cellular starvation induces translational shutdown as a survival strategy during energy crisis. However, specific mRNAs need to be translated to enable the production of stress response proteins. I propose that there are two potential functions of RNA ADP-ribosylation: (1) to allow translation of specific ADPr-mRNAs in an m7G cap-independent manner during starvation, or (2) to enable storage of ADPr-mRNAs for rapid m7G recapping and translation when starvation conditions subside. I will thus identify the mRNAs that are modified upon stress and determine how the ADPr cap influences their function in cells. In addition to determining the role of RNA ADP-ribosylation in response to nutrient stress, I aim to characterize the enzymes involved in the capping and removal of ADP-ribose from mRNA, utilizing a combination of inhibitors and cell lines with knocked-down or overexpressed enzymes. In summary, this project aims to determine the function of mRNA ADP-ribosylation in response to nutrient deprivation and to identify the relevant enzymes in cells.

• DFG Programme: Research Grants